PHLPP1 inhibits the growth and aerobic glycolysis activity of human ovarian granular cells through inactivating AKT pathway

Background Polycystic ovary syndrome (PCOS) is a disorder characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphologic features, and PCOS is associated with infertility. PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) has been shown to regulate AKT. The aim of present study is to investigate the role of PHLPP1 in PCOS. Methods The expression levels of PHLPP1 in dihydrotestosterone (DHT)-treated human ovarian granular KGN cells were determined by qRT-PCR and Western blot. PHLPP1 was silenced or overexpressed using lentivirus. Cell proliferation was detected by CCK-8. Apoptosis and ROS generation were analyzed by flow cytometry. Glycolysis was analyzed by measuring extracellular acidification rate (ECAR). Results DHT treatment suppressed proliferation, promoted apoptosis, enhanced ROS, and inhibited glycolysis in KGN cells. PHLPP1 silencing alleviated the DHT-induced suppression of proliferation and glycolysis, and promotion of apoptosis and ROS in KGN cells. PHLPP1 regulated cell proliferation and glycolysis in human KGN cells via the AKT signaling pathway. Conclusions Our results showed that PHLPP1 mediates the proliferation and aerobic glycolysis activity of human ovarian granular cells through regulating AKT signaling.


Background
Infertility is the inability to get pregnant after 1 year of regular intercourse [1].Infertility has a 9 to 18% prevalence [2].In USA, approximately 13% of reproductive age women seek treatment for infertility every year [3].About 85% of cases are because of ovulatory dysfunction, male factor infertility, and tubal disease.The other 15% of cases have "unexplained infertility" [3].Polycystic ovary syndrome (PCOS), a common metabolic disorder in premenopausal women, is characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphologic features [4,5].PCOS is a major cause of hyperandrogenism and infertility [6].The prevalence of infertility in women with PCOS ranges from 70 to 80% [7].PCOS affects 6-20% of reproductive aged women [8].The etiology is still unclear, but data support that it may be a multigenic disorder with strong epigenetic influences [4].Risk factors include genetics, neuroendocrine, lifestyle/environment, obesity [9].Treatment of PCOS is individualized accordingly.For example, weight control should be considered for overweight patients, clomiphene is recommended for infertility [10].Regardless of the advances in treating PCOS, more studies are needed for better understanding the underlying mechanisms in order to provide more effective and precise treatment.
Glycolysis metabolizes glucose to pyruvate and generates ATP in cytoplasm [11].The 3 key enzymes for glycolysis are hexokinase, phosphofructokinase, and pyruvate kinase [12].Glycolysis plays a very important role in various pathological processes.For instance, Lim et al. have reported that inhibiting glycolysis and EGFR effectively suppressed mammary cancer growth [13].Xiang et al. demonstrated that enhanced glycolysis in skeletal muscle coordinates with adipose tissue in systemic metabolic homeostasis [14].Glycolysis is also essential for sperm motility and male fertility because sperm require ATP that is produced by mitochondrial oxidative phosphorylation and glycolysis to provide energy for motility [15].Aberrant expression of glycolytic enzymes has been shown to be associated with infertility [16].Glycolysis also involves in PCOS.For example, Zhao et al. have demonstrated that elevated glycolysis and decreased tricarboxylic acid cycle were presented in women with PCOS [17].Dysregulation of glycolysis has also been associated with PCOS patients with hyperplasia [18].Despite the remarkable success of the study of glycolysis, the exact role of glycolysis in PCOS and how glycolysis is involved remain unclear and need to be elucidated.
PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) is a new member of the phosphatome [19].PHLPP1 was first discovered by Gao et al. for its inhibiting growth factor signaling through dephosphorylation of AKT (protein kinase B, PKB) [20].Dysregulation of PHLPP involves in different pathophysiology, from cancer to metabolic diseases [21].Moc et al. have reported that physiological activation of AKT signaling by PHLPP1 deletion protects against pathological hypertrophy [22].In contrast, overexpressing PHLPP1 decreases Akt and suppresses melanoma cell growth [23].It is known that the phosphoinositide 3-kinase-AKT pathway regulates glycolysis [24].AKT activation leads to promotion of aerobic glycolysis and overexpressing AKT leads to increased glycolytic rate [25,26].As mentioned above, dysregulated glycolysis plays a crucial role in PCOS.However, the role of PHLPP1/AKT/glycolysis in infertility and PCOS is not clear and remains to be elucidated.Therefore, how PHLPP1 regulates glycolysis and how this PHLPP1-regulated-glycolysis affects human ovarian granular cell proliferation were investigated in this study to provide scientific basis for developing new treatments for PCOS.

Cell proliferation
Cell Counting Kit-8 (CCK-8; Dojindo, Japan) was used to detect cell proliferation.Briefly, KGN cells were planted into 96-well culture plate with a primary density of 3 × 10 3 cells per well.At the specified time points, CCK-8 solution was added into the cells at 37 °C for 2 h incubation, and the absorbance was scanned by a microplate reader.

Flow cytometry assay
The staining procedures followed two steps: (1) 20 min of incubation with Annexin-V-FITC at 4 °C and (2) another 20 min of incubation with propidium iodide (PI).A flow cytometer (BD Biosciences) was used to measure apoptosis.

ROS production
Intracellular ROS accumulation was measured via 2' ,7'-diclorodihydrofluorescein diacetate (DCFDA) staining.Briefly, KGN cells were incubated with 10 µM DCFDA in PBS for 25 min at 37 °C, and nuclei were stained with DAPI for 10 min.The cells were collected and analysed using a flow cytometer.

Glycolysis assay
Extracellular acidification rate (ECAR) was measured with a XF24 Extracellular Flux Analyzer [27].Briefly, cells digested to a density of 1 × 10 4 /well, were seeded in XF-24 culture plates (Agilent Technologies, Santa Clara, CA, USA), and were then placed in an incubator of 37 °C and 5% CO 2 for 24 h.Around 1 h before detection, culture medium was replaced by XF Base Medium (Agilent Technologies).Subsequently, the cells were treated sequentially with 1 µM of glucose, 1 µM of oligomycin (ATP synthase inhibitor) and 0.5 µM of 2-DG (the glycolytic inhibitor) at time points for measurement of ECAR.

Sample collections
This study was approved by the Ethics Committee of Affiliated Hospital of Inner Mongolia Minzu University.Informed consents were obtained from women ages 25 to 38 years old.Granulosa cells were collected from 30 PCOS women [15 hyperandrogenic PCOS (HA-PCOS), 15 normo-androgenic PCOS (NA-PCOS)] and 15 controls who had normal endocrine as previously described [28].Demographic and clinical parameters of participants were listed in Table 1.

Statistical analysis
GraphPad Prism8.4.2 was used for data analysis.Data was expressed as mean ± SD.Comparisons between 2 groups were performed by T-test, comparisons among multiple groups were performed by one-way ANOVA.p-values < 0.05 indicate statistical significance.

DHT treatment inhibited the proliferation and glycolysis activity in human KGN cells
DHT was used to detect the action of high levels of androgen on proliferation and glycolysis activity in KGN cells.DHT treatment significantly suppressed KGN cell proliferation (Fig. 1A), and promoted the apoptosis of human KGN cells (Fig. 1B).DHT treatment also significantly inhibited glycolysis (Fig. 1C), and elevated ROS generation (Fig. 1D) in KGN cells.These data suggest that DHT treatment inhibited the proliferation and glycolysis of KGN cells.

Knockdown of PHLPP1 ameliorated the DHT-induced injury in human KGN cells
As mentioned above, PHLPPs are important regulars of glycolysis and AKT.To figure out whether PHLPPs play a role in DHT regulation of cell proliferation and glycolysis, the expression of PHLPPs was examined after DHT treatment.Results showed that DHT treatment time-dependently up-regulated PHLPP1 at both mRNA and protein levels, but did not affect PHLPP2 (Fig. 2A  and B).Similarly, it was found that the mRNA and protein levels of PHLPP1 were also increased significantly in the HA-PCOS subjects compared with the controls or NA-PCOS subjects (Fig. 2C and D).Therefore, PHLPP1 was focused in the subsequent study.To study the role of PHLPP1 in DHT regulation of cell proliferation and glycolysis, lentivirus was used to silence PHLPP1.Results showed that PHLPP1 was successfully silenced (Fig. 3A and B).More importantly, silencing PHLPP1 significantly ameliorated DHT-suppressed proliferation of KGN cells (Fig. 3C).DHT treatment promoted apoptosis of KGN cells, which was abolished by silencing of PHLPP1 (Fig. 3D and E).The inhibitory effect of DHT on glycolysis was significantly suppressed by PHLPP1 knockdown (Fig. 3F).ROS levels were significantly elevated by DHT treatment, but silencing of PHLPP1 remarkably decreased ROS levels in KGN cells (Fig. 3G).These results demonstrate that knockdown of PHLPP1 ameliorated the DHT-induced injury in human KGN cells.

Inhibiting AKT rescued the effect of PHLPP1 silencing on cell proliferation and glycolysis in DHT-treated KGN cells
To figure out whether AKT signaling pathway play a role in DHT regulation of cell proliferation and glycolysis, the activation of AKT was examined after DHT treatment.Results showed that DHT treatment timedependently down-regulated activation of AKT signaling pathway (Fig. 4A).To further investigate the role of AKT signaling, AKT inhibitor, MK-2206 was introduced.Administration of MK-2206 abolished DHTsuppressed proliferation of KGN cells with PHLPP1 silencing (Fig. 4B).MK-2206 also inhibited the glycolysis activity in DHT-treated PHLPP1-silencing KGN cells (Fig. 4C).Western blotting results indicated that MK-2206 significantly decreased levels of p-AKT induced by DHT and PHLPP1 silencing (Fig. 4D).Together, these results suggest that inhibiting AKT rescued the effect of PHLPP1 silencing on cell proliferation and glycolysis in DHT-treated KGN cells.

AKT activator SC79 disrupted the effect of PHLPP1 overexpression on cell proliferation and glycolysis in KGN cells
To further study PHLPP1 and AKT, PHLPP1 was successfully overexpressed (Fig. 5A and B), and an AKT activator, SC79, was introduced.Results demonstrated that administration of AKT activator, SC79, significantly increased proliferation of KGN cells with PHLPP1 overexpression (Fig. 5C).Administration of SC79 also remarkably increased glycolysis activity of KGN cells with PHLPP1 overexpression (Fig. 5D).Western blotting results indicated that SC79 significantly increased level of p-AKT in KGN cells with PHLPP1 overexpression (Fig. 5E).These data suggest that the AKT activator, SC79, disrupted the effect of PHLPP1 overexpression on cell proliferation and glycolysis in KGN cells.

Discussion
We demonstrated that DHT treatment suppressed proliferation, promoted apoptosis and ROS, and inhibited glycolysis of KGN cells, which were ameliorated by silencing PHLPP1.Mechanism study indicated that DHT-caused injury in PHLPP1-silencing cells was abolished by administration of AKT inhibitor.In contrast, administration of AKT activator disrupted the function of PHLPP1.For the first time, our study indicated that PHLPP1 mediates cell proliferation, apoptosis, ROS generation, and glycolysis of KGN cells by regulating AKT signaling pathway (Fig. 5F).
Converting glucose to pyruvate, glycolysis involves in a many bioprocesses [11].Glycolysis plays a fundamental role in supporting cell growth and proliferation [12].More importantly, it has been reported that perturbation of the expression of glycolytic enzymes was associated with infertility [16].Zhao et al. have reported that enhanced glycolysis and inhibited tricarboxylic acid cycle were found in women with PCOS [17].Here, we found that DHT administration resulted in significant damage of KGN cells through regulating PHLPP1 expression.Data demonstrates, for the first time, that silencing PHLPP1 ameliorated DHT-induced cell injury.
AKT plays a very important role in cell growth, division, and apoptosis [29].AKT activation increases glycolysis [25].Hojlund et al. have shown that AKT activation was significantly inhibited in PCOS skeletal muscle [30].These data suggested that the status of AKT pathway might be tissue or cell-type specific.Our study indicated that inhibition of AKT rescued the role of PHLPP1 silencing in DHT-treated KGN cells, while activation of AKT disrupted the function of PHLPP1 overexpression.The findings suggest that PHLPP1 regulates glycolysis and cell proliferation through regulating AKT, and indicate that targeting AKT/PHLPP1 might provide a new direction for manipulating glycolysis and cell proliferation.
PHLPP1, first discovered by Gao et al., suppresses growth factor signaling through dephosphorylating Akt [20].Overexpression of PHLPP1 has been shown to inhibit cell proliferation [23], while silencing of PHLPP1 has been shown to contribute to cell growth through regulating of AKT phosphorylation [23].Studies also supported that AKT activation promoted glucose uptake and aerobic glycolysis [25,26].In the current study, we revealed a role of PHLPP1, showing that PHLPP1 suppressed glycolysis and promoted apoptosis of KGN cells through inhibition of AKT signaling pathway.Our study further showed PHLPP1 overexpression-caused cell injury could be abolished by administration of AKT activator, SC79.The results improved the understanding of the possible mechanism by which PHLPP1 regulates the proliferation and glycolysis of KGN cells.Previous studies have demonstrated that glycolytic enzymes are regulated by complex mechanisms, such as AKT, ERK, and NF-κB pathways [31][32][33].The AKT pathway is responsible for glycolysis by regulating HIF-1α target genes ENO1 and LDHA [34,35].These data indicate that PHLPP1 may regulate ENO1 and LDHA expression via the AKT/HIF-1α signaling pathway.Xiong et al. demonstrated that PHLPP1 suppresses glycolysis via inhibiting Akt phosphorylation and mitochondrial localization of HK2, a key enzyme that determines the direction and magnitude of glucose flux [36].However, whether other glycolytic enzymes and signlaing pathways are regulated by PHLPP1 should be further determined.It is worth to mention that only in vitro experiments and human ovarian granular KGN

Conclusions
Taken together, the results revealed a novel role of PHLPP1/AKT axis, indicating that knockdown of PHLPP1 ameliorated the DHT-induced injury in human KGN cells through regulation of AKT signaling pathway.The data highlighted the significance of PHLPP1/AKT axis which may benefit the treatment of PCOS.
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Fig. 1
Fig. 1 DHT treatment inhibited the proliferation and glycolysis activity in KGN cells.A Proliferation of KGN cells was deeply decreased after DHT treatment.B DHT treatment significantly increased apoptosis of KGN cells.C The glycolysis activity of KGN cells was suppressed by DHT treatment.D DHT treatment significantly promoted the production of ROS in human KGN cells.* p < 0.05, *** p < 0.001 vs. Vehicle

Fig. 2 Fig. 3
Fig. 2 PHLPP1 is upregulated in DHT-treated KGN cells and patients with PCOS.A, B mRNA and protein levels of PHLPP1 and PHLPP2 in KGN cells after 6, 12 and 24 h of DHT treatment.C, D mRNA and protein levels of PHLPP1 in patients of the control, HA-PCOS, and NA-PCOS groups.** p < 0.01,*** p < 0.001 vs. Vehicle or control.!!! p < 0.001 vs. NA-PCOS

Table 1
Basic clinical features of participants BMI Body mass index, SHBG Sex hormone-binding globulin, FAI Free androgen index, DHEAS Dehydroepiandrosterone sulfate, 17-OHP 17-Hydroxyprogesterone, FSH Follicularstimulating hormone, LH Luteinizing hormone, TSH Thyroid-stimulating hormone a Significant difference between control and NA-PCOS or HA-PCOS subjects (p < 0.001) b Significant difference between NA-PCOS and HA-PCOS subjects (p < 0.001)